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Super-Resolution Microscopy Approaches for Live Cell Imaging View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biophysical Journal Volume 107 October 2014 1777–1784 1777 Biophysical Review Super-resolution Microscopy Approaches for Live Cell Imaging Antoine G. Godin,1,2 Brahim Lounis,1,2 and Laurent Cognet1,2,* 1University of Bordeaux, LP2N, UMR 5298, Talence, France; and 2Institut d’Optique Graduate School and Centre National de la Recherche Scientifique, LP2N, UMR 5298, Talence, France ABSTRACT By delivering optical images with spatial resolutions below the diffraction limit, several super-resolution fluores- cence microscopy techniques opened new opportunities to study biological structures with details approaching molecular structure sizes. They have now become methods of choice for imaging proteins and their nanoscale dynamic organizations in live cells. In this mini-review, we describe and compare the main far-field super-resolution approaches that allow studying endogenous or overexpressed proteins in live cells. INTRODUCTION The decryption of cell functions and subcellular processes has using saturable optical processes that deexcite emitters constantly benefited from advances in microscopy. In partic- formerly excited by a focused laser beam. These processes ular, the developments of fluorescence microscopy and of work to prevent fluorescence emission from specific regions numerous fluorescent probes allowing the study of specific of the excitation beam by driving the molecules in these biomolecules at work in their native environment were instru- regions between bright and dark states using a depletion light mental to the advance of live cell mechanism investigations. beam. One elegant and efficient strategy consists of using The optical resolution of microscopes is limited by the diffrac- stimulated emission by a high-intensity (>MW/cm2), tion of light, which commonly sets a limit of ~l/2 in far-field doughnut-shaped laser beam superimposed with the focused microscopy. By delivering optical images with spatial resolu- excitation laser beam, completely preventing fluorescence tions below the diffraction limit, super-resolution fluorescence emission from emitters in peripheral regions of the excitation microscopy offered new promises to study molecular pro- beam. This process was coined ‘‘stimulated emission deple- cesses with greater detail than with conventional microscopies tion’’ (STED) (3). A doughnut-shaped depletion beam is the (1,2). Most of these methods rely on the control of the number simplest design; however, in general, any depletion beam of emitting molecules in specific imaging volumes. This can featuring a spatial intensity distribution with one or several be achieved by controlling local emitter fluorescent state intensity zeroes can be used to perform STED images. populations or the labeling densities of fluorescing probes at To generate a super-resolved image with STED based on any given time during the image acquisition process. In this local excitation volumes, one must scan the excitation/deple- mini-review, we will discuss the key features of super-resolu- tion effective volumes over the sample in a deterministic tion techniques used for live-cell studies. We schematically point-by-point manner or by use of parallelized scanning divide them into three major groups: those based on highly schemes (4,5). STED was successfully applied in several localized fluorescence emission volumes; those based on live samples to study slow morphing and movements of structured illumination; and those based on single-molecule organelles such as reticulum endoplasmic or microtubules localizations. A didactic representation of the three families (6), subcellular organization in live cells (7), and synaptic of super-resolution approaches is presented in Fig. 1. structures in live samples (7–9). For live cell studies, one should bear in mind that relatively high laser powers are needed in STED, especially when using continuous wave SUPER-RESOLUTION BASED ON HIGHLY laser beams (e.g., ~MW/cm2 (10)). Using pulsed excitation LOCALIZED FLUORESCENCE EMISSION beam together with time-gating detection allowed a ~2–3- VOLUMES fold reduction in laser power (11). In addition, photobleach- Stimulated emission depletion (STED) and ing is a limiting factor for long-term live sample imaging reversible saturable optical fluorescence because each fluorescent molecule undergoes a large num- transition (RESOLFT) ber of exciting/de-exciting cycles in the depletion beam. An approach similar to STED using much lower inten- In a far-field confocal microscope, the effective fluorescence sities to deplete emitting molecular levels (~kW/cm2)(12) volume can be reduced below the diffraction limit (3)by is based on reversible photoswitching of marker proteins between a fluorescence-activated and a nonactivated state Submitted June 18, 2014, and accepted for publication August 7, 2014. (13–15), whereby one of the transitions is accomplished *Correspondence: [email protected] by means of a spatial intensity distribution featuring a Editor: Brian Salzberg. Ó 2014 by the Biophysical Society 0006-3495/14/10/1777/8 $2.00 http://dx.doi.org/10.1016/j.bpj.2014.08.028 1778 Godin et al. C RESOLFT/STED A Object Excitation over the sample Depletion Scanning laser beams D Structured illumination microscopy (SIM) 3 patterns*3 modulations = 9 images Software B Diffraction Reconstruction limited image E Single molecule approaches Acquired Images ... ... Image Reconstruction from localizations Single molecule localization FIGURE 1 Schematic description of the superresolution microscopy approaches. All images for this didactic description are computer-generated. Object to be imaged consisted of fluorescent emitters (A) and corresponding diffraction-limited image (B). (C) In RESOLFT/STED, a focused excitation beam (cyan) superimposed with a doughnut-shaped depletion beam (red) are scanned over the sample to acquire an image at high resolution (down to ~50– 80 nm in live cells). (D) In SIM, after the required software reconstruction, multiple wide-field images are acquired using sinusoidal illumination grid patterns to obtain high-resolution images (down to ~50–100 nm in live cells using nonlinear saturated illumination). (E) In single-molecule localization microscopy, a large number of wide-field images containing a few isolated single fluorescent emitters are successively acquired. A high-resolution image is reconstructed from the localizations of each individual molecule. Resolutions down to ~50 nm are commonly achieved in live cells. In the example provided, we considered the detection of 80% of the molecules present in the object image. Scale bar represents 1 mm. To see this figure in color, go online. zero. This generalized approach was named after ‘‘revers- vides only an approximately twofold resolution enhance- ible saturable optical fluorescence transition’’ (RESOLFT). ment of standard wide-field microscopy as compared to Bright photostable switchable fluorophores and fluorescent other super-resolution methods (19). Nonlinear saturated proteins development were particularly instrumental in the SIM using fluorophore saturation or photoswitchable pro- development of these techniques (14–16). Importantly, fluo- teins as in RESOLFT can achieve higher resolution rescent proteins provide specific 1:1 protein labeling and enhancement (~50 nm), but requires an increased number offer the possibility of intracellular live cell imaging. of image acquisitions (up to 63) and a complex reconstruc- tion process (20,21). SIM has been demonstrated for long- term, live cell imaging in microtubules and other dynamic STRUCTURED ILLUMINATION MICROSCOPY (SIM) structures (21–23). Three-dimensional SIM imaging has Structured illumination microscopy (SIM) is based on stan- been further achieved using 15 different pattern acquisitions dard wide-field microscopy and is compatible with most per axial planes for reconstruction instead of nine images to standard fluorophores and labeling protocols. It uses reject the out-of-focus light (24). Whole-cell volume imag- nonuniform illuminations with known spatial patterns ing has been performed using three-dimensional SIM in two (e.g., originally a sinusoidal grid, but other illumination dis- colors (25). And, interestingly, fast SIM imaging (11 Hz) tributions can also be used (17)). From multiple acquisitions has even been developed with a 100-nm resolution for a (e.g., nine images, incorporating three phase shifts for three small field of view (~8 Â 8 mm2)(18). pattern orientations (18)), high spatial frequency informa- tion is retrieved with a dedicated algorithm, comprising a SINGLE-MOLECULE LOCALIZATION method inaccessible with standard illumination schemes MICROSCOPY APPROACHES (19). Contrary to standard laser scanning modalities like STED/RESOLFT, SIM allows acquisition of a large field It is well known that the position of isolated single fluores- of view over limited times. However, SIM routinely pro- cent emitters can be determined by image analysis with Biophysical Journal 107(8) 1777–1784 Live Cell Super-resolution Imaging 1779 greater precision than is available from the diffraction limit with the use of reducing/oxidizing buffers that can affect alone. This feature, which has been used for more than 20 cell integrity (41). Of special interest is that STORM has years in live cell, single-particle/-molecule studies (26), is been shown to take advantage of some reduction in thiol key to providing today’s super-resolved images. Super-res-
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